CDK8 has been identified as being frequently upregulated in various types of human cancer such as colorectal cancer, certain sub-types of breast cancer and acute myeloid leukemia. In different tumor cell lines RNA interference mediated knockdown of CDK8 results in significant inhibition of tumor cell growth in vitro and in vivo. CDK8, therefore, represents an interesting target for the development of novel anti-cancer drugs. Various compounds targeting CDK8/Cyclin C with high potency in low nanomolar range have been reported. These compounds differ with respect to their binding modes, but all of them show high potency in biochemical assays, inhibition of tumor cell growth, and for many in vivo activities in different tumor cell xenograft models have been demonstrated. CDK8 binds to Cyclin C resulting in the active protein kinases complex. CDK8/Cyclin C interacts with MED12 and MED13, two proteins that regulate CDK8/Cyclin C activity and its interaction with the mediator complex. Mediator consists of 26 subunits and the interaction of the CDK8/Cyclin C/MED12/Med13 complex with mediator enables CDK8/Cyclin C to regulate gene transcription via C-terminal phosphorylation of RNA-polymerase II. This mechanism mediates an indirect regulation of transcription factors such as ß-catenin. In addition of its function via phosphorylation of RNA-polymerase II, CDK8/Cyclin C can also directly regulate the activity of transcription factors by phosphorylation. Best known examples are STAT1 and STAT5 which are phosphorylated by CDK8/Cyclin C on serine 727 and Serine 726, respectively. Recent data indicates that mutations of MED12 can result in a reduced sensitivity of CDK8/Cyclin C towards certain published CDK8/Cyclin C inhibitor in a cellular context. In this study we performed real-time quantitative PCR (qPCR) analyses to characterize the effect of different type I and type II CDK8/Cyclin C inhibitors on the cellular expression of selected genes controlled by two CDK8/Cyclin C dependent pathways, namely the WNT/ß-catenin- and the STAT-pathway. Furthermore, results of qPCR analyses of tumors derived from human xenograft in vivo models treated with type I and type II CDK8/Cyclin C inhibitors will be presented. Citation Format: Laura M. Jordt, Frank Totzke, Joachim Lauterwasser, Jan E. Ehlert, Koen Hekking, Bas Aerts, Cynthia Obodozie, Holger Weber, Gerhard Müller, Michael H. Kubbutat. Real-time quantitative PCR based analysis of transcriptional effects of CDK8/Cyclin C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2318.

The family of human Cyclin dependent kinases (CDKs) comprises 20 different CDKs that play critical roles in the regulation of cell cycle progression, gene transcription and neuronal function. Deregulation of different CDKs is frequently observed in human cancer. Enzymatic kinase activity of CDKs is dependent on the binding of a member of the Cyclin protein family. So far more than 15 Cyclins have been described most of them can bind and activate different CDKs. Current data suggest the physiological relevance of at least 50 different CDK/Cyclin complexes. Since the 20 CDKs share significant structural homology and regulate different function in cell growth and development, selectivity of compounds within the CDK family is of critical importance. Approval of a first CDK inhibitor (Palbociclib) targeting CDK4/6 for the treatment of ER+/HER+- breast cancer served as a clinical proof that targeting specific members of the CDK protein kinase family is a versatile approach to treat cancer. The approval of the first CDK inhibitor sparked the research and development of other inhibitors targeting different members of the CDK-family. Currently, four additional CDK4/6 inhibitors have been approved and more than 15 CDK inhibitors with limited selectivity are in different preclinical or clinical development phases. However, the critical importance of selectivity within the CDK family is underlined by the fact that the clinical development of four CDK9 inhibitors has been stopped due to the lack of selectivity and high toxicity. Different approaches (including inhibition by covalent binding) have resulted in more selective inhibitors, especially against CDKs like CDK7, CDK9 and CDK12. Although several biochemical activity assays for different CDKs have been set up and used for selectivity testing, so far no panel covering all 20 human CDKs using one assay technology has been described. We report here the setup of a biochemical in-vitro activity assay panel of 32 CDK/Cyclins complexes generated recombinantly in insect cells covering the complete set of all 20 human CDKs. For all 32 complexes a radiometric biochemical activity assay has successfully been established allowing to characterize inhibitors with respect to their biochemical selectivity applying the same assay technology. Using the comprehensive CDK panel we determined the IC50 values of more than 15 CDK inhibitors that have been either approved or are in different preclinical and clinical development phases. Results will be presented showing the selectivity of these inhibitors not only for all 20 CDKs but also for specific CDKs forming active complexes with two or more different cyclins. This CDK screening panel allows the generation of comparative data on compound selectivity early in development, thereby helping to reduce the risk of designing compounds with suboptimal target selectivity. Citation Format: Daniel Müller, Frank Totzke, Thomas Weber, Andreas Gericke, Diane Krämer, Carolin Heidemann-Dinger, Constance Ketterer, Michael H. Kubbutat. Comprehensive characterization of CDK inhibitors using a complete panel of all 20 human cyclin-dependent kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2304.

Leucin-rich repeat kinase 2 (LRRK2) plays an important role in the onset of sporadic as well as familial Parkinson’s disease. Pathogenic gain-of-function mutations of LRRK2 are associated with aberrant LRRK2 hyperactivity which results in neurotoxicity and protein aggregation caused by dysfunctional autophagy and vesicle trafficking. Thus, the development of LRRK2 inhibitors represents a promising strategy for the treatment of Parkinson’s disease. Interestingly LRRK2 mutations have also been reported to increase the risk for the onset of different types of cancer (e.g. breast, thyroid, lung). Several studies suggest that LRRK2 is involved in the regulation of different cancer-related pathways (e.g. ATM-p53-p21-pathway, JNK pathway). Deregulation of LRRK2 activity caused by mutations has been shown to interfere with these pathways thereby increasing the risk to develop certain types of cancer. Since anti-cancer treatments mostly target the same pathways, we hypothesized that LRRK2 inhibitors may affect anti-cancer treatments in specific cancer cell types. In this study we report on the analyses of various inhibitors (e.g. MLi-2 and PF-06447475) on LRRK2 autophosphorylation at S935 in a cellular phosphorylation assay using the non-small cell lung cancer cell line A549 in comparison to biochemical LRRK2 activity assays. Furthermore, we compare the direct LRRK2 inhibitor effect on the proliferation of 140 cell lines, as well as their potential combinatorial impact on the potency of chemotherapeutic agents (e.g. Adriamycin). The observed effects can help to understand the implications of pharmaceutical LRRK2 inhibition in the treatment of both Parkinson’s disease and cancer. Citation Format: Franziska Fimm-Todt, Joachim Lauterwasser, Eva-Maria Egenter, Christian Weber, Daniel Feger, Katharina Schaich, Sarah Ulrich, Oliver Siedentopf, Frank Totzke, Michael Kubbutat, Jan Erik Ehlert. Cytotoxic effects of LRRK2 inhibitors in combined treatment with chemotherapeutic agents on a large panel of cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4072.

Cyclin dependent kinases (CDK) form a family of 20 different CDKs with an additional 6 CDK-like proteins. Members of this family have been shown to be involved in the regulation of many critical cellular processes, including the regulation of cell cycle progression, gene transcription and neuronal function. Deregulated CDK activity has been observed frequently in human cancer and other diseases. Enzymatic kinase activity of CDKs has been found to depend on the formation of complexes with regulatory proteins of the Cyclin family and requires in some cases the presence of additional complex partners, like MAT1 for CDK7/Cyclin H. To date, at least 15 different Cyclins have been described. For some CDKs it has been shown that they can also be activated by binding to proteins distinct from classical Cyclins. However, even while the 20 canonical CDKs share significant structural homology, they regulate very different functions of cell growth and development, partially due to their inherent substrate specificity but also regulated by their respective Cyclin partners. During the past 30 years several CDKs have been subject to extensive study resulting in a multitude of publications. This includes specifically CDK1, CDK2 and CDK4/6 and to a lesser extend CDK7 and CDK9. Other CDKs have been studied much less extensively and consequently the accumulated knowledge about their biological function and regulation is very limited. CDK15 (also known as PFTK2 or ALS2CR7) is one of these understudied members of the CDK family, with less than 20 publications in PubMed and the search term "CDK15" in contrast to more than 8600 hits for "CDK2". Its biological function is largely obscure even while evidence was presented that CDK15 is involved in the regulation of breast and colorectal cancer. Comparable to all other members of the CDK family, recombinantly overexpressed purified CDK15 does not exhibit detectable in-vitro activity and to date no Cyclin or other activating complex partner for CDK15 has been published. Since CDK inhibitors have been in the focus of interest at least since the approval of the first candidate (Palbociclib) for the treatment of ER+/HER+ breast cancer, testing the selectivity of other drug candidates within the complete group of CDKs is critically important to avoid unwanted off-target effects and toxicity. In order to enable in-vitro activity studies with CDK15 we evaluated different CDK15 constructs in combination with a comprehensive panel of different Cyclins using the baculo virus expression system. CDK15 and the respective Cyclins were co-expressed in insect cells, purified by affinity chromatography and tested for in-vitro activity using a panel of generic, broad-spectrum protein kinase substrates. We will present data on the identification of CDK15 activating Cyclins and the biochemical characterization of the in-vitro kinase activity of such CDK15/Cyclin complexes including a panel of kinase inhibitors. Citation Format: Daniel Mueller, Constance Ketterer, Diane Kraemer, Carolin Heidemann-Dinger, Lena Pilgermayer, Thomas Weber, Andreas Gericke, Frank Totzke, Michael H. Kubbutat. Identification of CDK15 activating cyclins by a recombinant co-expression approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5980.